Phosphorylcholine conjugates and corresponding antibodies

ABSTRACT

In subjects with hypertension, increases in intima-media thickness (IMT) at four years were less in subjects also having high autoantibodies particularly IgM, to phosphorylcholine. The presence or absence of autoantibodies, particularly IgM, against phosphrylcholine is thus related to an increased or decreased risk of developing ischemic cardiovascular diseases. A method to determining antibodies, particularly IgM antibodies, toward phosphorylcholine is proposed in this invention to identify subjects at risk of developing ischemic cardiovascular diseases. Animal experiments show that medium to high levels of antibodies, particularly IgM antibodies, can be detected in plasma after active immunization with a keyhole limpet hemocyanin (KLH)-phosphorylcholine conjugate. Pharmaceutical compositions comprising a phosphorylcholine conjugate or antibody preparations, for example a monoclonal antibody, with specificity to a phosphorylcholine conjugate is proposed as is use of these compositions as active or passive immunogens in the treatment or prevention of atherosclerosis.

This application is a continuation of U.S. application Ser. No.14/155,903, filed Jan. 15, 2014, which is a divisional of U.S.application Ser. No. 13/208,138, filed Aug. 11, 2011, which is adivisional of U.S. application Ser. No. 10/599,934, now U.S. Pat. No.8,012,483, filed May 31, 2007, as a national phase application under 35U.S.C. §371 of International Patent Application No. PCT/GB2005/001463filed Apr. 15, 2005, which claims priority to U.S. Provisional PatentApplication No. 60/521,384 filed Apr. 15, 2004. The entire texts of theabove-referenced disclosures are specifically incorporated herein byreference without disclaimer.

FIELD OF THE INVENTION

This invention relates to the field of treatment and risk assessment foratherosclerosis and ischemic cardiovascular diseases.

DESCRIPTION OF RELATED ART

Atherosclerosis is a chronic disease that causes a thickening of theinnermost layer (the intima) of large and medium-sized arteries. Itdecreases blood flow and may cause ischemia and tissue destruction inorgans supplied by the affected vessel. Atherosclerosis is the majorcause of cardiovascular disease including myocardial infarction, strokeand peripheral artery disease. It is the major cause of death in thewestern world and is predicted to become the leading cause of death inthe entire world within two decades.

The disease is initiated by accumulation of lipoproteins, primarilylow-density lipoprotein (LDL), in the extracellular matrix of thevessel. These LDL particles aggregate and undergo oxidativemodification. Oxidized LDL is pro-inflammatory, toxic and causesvascular injury. Atherosclerosis represents in many respects a responseto this injury including inflammation and fibrosis.

In 1989 Palinski and coworkers identified circulating autoantibodiesagainst oxidized LDL in humans. This observation suggested thatatherosclerosis may be an autoimmune disease caused by immune reactionsagainst oxidized lipoproteins. At this time several laboratories begansearching for associations between antibody titers against oxidized LDLand cardiovascular disease. However, the picture that emerged from thesestudies was far from clear. Antibodies existed against a large number ofdifferent epitopes in oxidized LDL, but the structure of these epitopeswas unknown. The term “oxidized LDL antibodies” thus referred to anunknown mixture of different antibodies rather than to one specificantibody.

It is well established that there is an ongoing inflammation in theatherosclerotic lesions, characterized by activation of immune competentcells and production of inflammatory cytokines. Established risk factorslike hypertension, blood lipids, diabetes and smoking are likely topromote this inflammatory reaction, but the mechanism by which thisoccurs are not well characterized and different non-mutually exclusivepossibilities exist. Several different autoantigens that could elicitthis immune reactivity have been proposed, including oxidized lowdensity lipoprotein (oxLDL) and heat shock proteins (HSP)^(2,3).Available data on the role of immune reactions in atherosclerosisindicate a complex relationship. One example of this is immunization inanimal models to influence atherogenesis. When HSP is used,atherosclerosis increases but decreases when oxLDL is the antigen^(4,5).

The role of aOxLDL in human disease appears to be complex. In humans, ithas previously been demonstrated that aOxLDL is higher in healthycontrols than in men with borderline hypertension, an example of earlycardiovascular disease⁶. Recent studies are in line with thisobservation^(7,8). On the other hand, several authors have reported thataOxLDL are raised in human cardiovascular diseases (CVD), especially atlater stages^(2,3,9,10). One example is systemic lupus erythematosus(SLE) and autoimmune disease associated with a very high risk of CVD.SLE-patients with a history of CVD had clearly raised aOxLDL levels¹¹.These to some extent contradictory results may depend on differentmethods and stages of LDL-oxidation, yielding differences inantigenicity. It is also likely that disease stage and risk factorprofile are related to antibody levels.

Oxidized low density lipoprotein (oxLDL) itself has many proinflammatoryproperties including activation of T cells^(12,13),monocytes/macrophages and endothelial cells¹⁴⁻¹⁶. OxLDL promotesinflammation also in immune competent cells from atheroscleroticlesions¹⁷. However, it should be noted that oxLDL may also ameliorateacute inflammatory reactions and instead promote a more low-gradechronic inflammation as that seen in atherosclerosis¹⁸. It isinteresting to note that many biological effects of oxLDL are caused byplatelet activating factor (PAF)-like lipids in oxLDL¹⁹⁻²¹.

Phosphorylcholine (PC) is a major component not only in inflammatoryphospholipids like platelet activating factor-PAF (where it is essentialfor interaction with the PAF-receptor) and in oxLDL, but is also as animmunogenic components of many bacteria including S. pneumoniae ²².Furthermore, PC is expressed by apoptotic cells^(2,23).

In U.S. Pat. No. 5,455,032 phosphocholine conjugates have been used invaccines for inducing immunoprotection against infections such asStreptococcus pneumoniae. In a recent study²⁴ by Binder et al onpneumococcus vaccine in mice, it was also shown that vaccinationdecreased atherosclerotic lesion formation. It was found that manyautoantibodies to oxLDL derived from atherosclerotic mice sharestructural identity with antibodies which protect against commoninfectious pathogens, including Streptococcus pneumoniae. The study, inmice, not humans, does not give any information about specificity, orthat IgM anti-phosphorylcholine antibodies are significantly moreimportant than corresponding IgG antibodies as a protecting factor inatherosclerosis. Furthermore, phosphorylcholine conjugates have not beenused in the pneumococcus vaccine.

In another study it was shown²⁵ that antiphosphorylcholine antibodylevels are elevated in humans with periodontal diseases. The conclusionis that phosphorylcholine is an important oral antigen associated withorganisms in the periodontal flora and that anti-PC antibody is elevatedas a consequence of periodontal disease. No information is given withregards to the antibodies and possible protection from or progression ofatherosclerosis.

A couple of documents (e.g. WO2002080954 and WO0168119) related toimmunization treatment of atherosclerosis have been published but theseare either based on the use of peptide fragments of apolipoprotein B orantibodies to alpha/beta chains of a T cell receptor. A method to detectatherosclerotic plaque (WO9908109) using monoclonal antibodies tooxidation-specific epitopes on lipoprotein has also been described. Thisis different from the method proposed in this invention where aphosphorylcholine conjugate is used to detect antibodies, eg IgM or IgGantibodies, in subject samples.

SUMMARY OF THE INVENTION

The invention relates to pharmaceutical compositions comprising aphosphorylcholine conjugate, or an antibody preparation, for example amonoclonal antibody, with specificity to a phosphorylcholine conjugate,and the use of these compositions in the treatment or prevention ofatherosclerosis, for example in the treatment, prevention or reductionof further progression of atherosclerosis Furthermore, the inventionalso relates to the use of phosphorylcholine conjugates or said antibodypreparation, for example monoclonal antibody to produce a pharmaceuticalcomposition optionally with an adjuvant. Furthermore the inventionrelates to diagnosing the presence or absence of antibodies, for exampleIgM or IgG antibodies, related to increased or decreased risk ofdeveloping ischemic cardiovascular diseases.

A first aspect of the invention provides the use of a pharmaceuticalcomposition comprising at least one phosphorylcholine conjugate, or anantibody preparation, for example a monoclonal antibody, withspecificity to a phosphorylcholine conjugate, in the manufacture of amedicament for immunization and treatment of mammals, including humans,against atherosclerosis or an atherosclerotic related disease. Themedicament is intended to provide immunization having immunogenic ortherapeutic properties against atherosclerosis.

A second aspect of the invention provides a method for immunization andtreatment of a mammal, including a human, against atherosclerosis or anatherosclerotic related disease, the method comprising the step ofadministering to the mammal a pharmaceutical composition comprising atleast one phosphorylcholine conjugate, or an antibody preparation, forexample a monoclonal antibody, with specificity to a phosphorylcholineconjugate. The pharmaceutical composition is intended to provideimmunization having immunogenic or therapeutic properties againstatherosclerosis.

By phosphorylcholine conjugate is meant a phosphorylcholine moietylinked to a carrier, preferably via a spacer. The structural elementphosphorylcholine may comprise a derivative of phosphorylcholine.Examples of suitable phosphorylcholine conjugates are described in U.S.Pat. No. 5,455,032, as noted above. For example, U.S. Pat. No. 5,455,032provides phosphorylcholine conjugates in which the phosphorylcholinemoiety is linked by a straight chain alkyl and an amide linkage to avariety of immunological carriers. The phosphorylcholine conjugate mayfor example be a human serum albumin (HSA)- or keyhole limpet hemocyanin(KLH)-phosphorylcholine conjugate or a bovine serum albumin(BSA)-phosphorylcholine conjugate (for example as described in theExamples). PC-BSA (Phosphorylcholine-Bovine Serum Albumin) can bepurchased from Biosearch Technologies, INC (Ca, USA). HSA-BSA-can beconjugated by a chemical procedure, for example the following procedure:

O-(4-aminophenyl phosphoryl)-choline (I) can be prepared fromO-(4-nitrophenyl-phosphoryl)-choline (Sigma N 5879) in quantitativeyield by reduction with hydrogen gas at 1 atm with 10% palladium atcharcoal as a catalyst, according to a procedure described by Chesebro,B. in Biochemistry 11, (1972) 766.

(I) can be coupled to HSA by means of EDC(1-ethyl-3-(3-dimethyl-aminopropyl)-carbodiimide) in MES buffer pH 4,essentially according to a procedure described by Padilla, N. D. et alin J. Immun. Methods 293 (2004) 1-11. The conjugated HSA can isolated bydialysis against buffered saline at pH 7.4.

The carrier can be, for example, a protein, lipid or polymer. Thecarrier can be latex beads, for example as described in the Examples.

The medicament may be intended for administration by injection.

A further aspect of the invention provides the use of one or more of thephosphorylcholine conjugates as defined in relation to the precedingaspects of the invention, in the manufacture of a pharmaceuticalcomposition, optionally in combination with an adjuvant, forimmunotherapy or therapy for the treatment of ischemic cardiovasculardiseases.

A further aspect of the invention provides a method of prophylactic ortherapeutic treatment of a mammal, which may be a human being, sufferingfrom atherosclerosis or facing the risk of developing ischemiccardiovascular disease, whereby a therapeutically effective amount of atleast one phosphorylcholine conjugate or an antibody preparation, forexample a monoclonal antibody, with specificity to a phosphorylcholineconjugate is administered.

The invention also relates to methods to determine the presence orabsence of antibodies, for example IgM or IgG antibodies, againstphosphorylcholine which are related to an increased or decreased risk ofdeveloping ischemic cardiovascular diseases.

A further aspect of the invention provides a method of diagnosing thepresence or absence of antibodies, for example IgM or IgG antibodies,related to increased or decreased risk of developing ischemiccardiovascular diseases, using a phosphorylcholine conjugate.

Thus, a further aspect of the invention provides the use of aphosphorylcholine conjugate in a method for assessing a patient's riskof developing or progression of ischemic cardiovascular disease in whichthe patient's levels of antibodies, for example IgM or IgG antibodies,reactive with the phosphorylcholine conjugate are assessed.

Phosphorylcholine conjugates are described above. The phosphorylcholinemay be linked to a carrier via a spacer. The carrier may be a protein,which may be KLH (keyhole limpet hemocyanin) or human serum albumin(HSA). The carrier may be latex beads.

The patient's levels of antibodies, eg IgM or IgG antibodies, reactivewith the phosphorylcholine conjugate may be assessed using animmunoassay. Examples of suitable immunoassays are described below andwill in any case be apparent to those skilled in the art.

It may be desirable to measure antibodies reactive with oxLDL or MD-LDLas well as measuring antibodies, eg IgM or IgG antibodies, reactive withthe phosphorylcholine conjugate.

It may alternatively or in addition be desirable to measure levels ofHSP70, HDL, TNF and/or HSP60 (as discussed in the Examples) as well asmeasuring antibodies, eg IgM or IgG antibodies, reactive with thephosphorylcholine conjugate.

DETAILED DESCRIPTION OF THE INVENTION

The examples disclosed below are provided only for the purpose ofillustrating the present invention and should not be considered as anylimitation of the scope as outlined in the appended claims. Documentreferred to herein are hereby incorporated by reference.

An example of a method to determine the presence or absence (or level)of IgM antibodies against phosphorylcholine which is related to anincreased or decreased risk of developing ischemic cardiovasculardiseases is described. Other methods known in the art can also be used.Similar methods may be used to determine the presence or absence (orlevel) of IgG antibodies against phosphorylcholine.

Methods to Determine the Presence or Absence of IgM Antibodies AgainstPhosphorylcholine

IgM antibodies to PC-BSA were determined by an enzyme-linkedimmunosorbent assay method.

A microtiter plate was coated with PC-BSA (10 μg/ml; for example fromBiosearch Technologies, INC (Ca, USA) in phosphate buffered saline(PBS). After washings with PBS, the plates were blocked with a 2% BSAsolution. Serum samples were diluted (1:30) in 0.2% BSA-PBS. Plates wereincubated overnight at 40° C. and washed. Alkaline phosphataseconjugated goat anti-human IgM (diluted 1:7000 in the sample buffer)were added at 100 ul/well and incubated at 40° C. overnight. Afterwashings, colour was developed by adding an alkaline phosphatasesubstrate and incubating the plates for 60 min at room temperature inthe dark. The absorbances were read in a spectrophotometer at 405 nm.

Different carriers and spacers for phosphorylcholine have been tested.The exemplified carriers are not limited to these. Other carriers suchas other proteins, lipids or polymers, such as latex beads which areknown in the art, may also be used. Carriers are discussed in U.S. Pat.No. 5,455,032, as noted above.

The IgM or IgG antibodies detected by a method of the invention may alsobind to phosphorylcholine (PC) present in PC-containing compounds inwhich PC is exposed, for example in lysophosphatidylcholine (lysoPC;see, for example, Kim et al, J Exp Med. 2002 Sep. 2; 196(5):655-65).Thus, a method of the invention may detect IgM or IgG antibodies thatbind to lysphosphatidylcholine.

Synthesis of a Phosphorylcholine Conjugate and Preparation of aPharmaceutical Composition

Latex beads (0.20 μm or 0.81 μm) were suspended in PBS and mixed overnight with a 10 μg/ml solution of phosphorylcholine-BSA. The beads werethen centrifuged and washed several times with buffer and blocked with a10 μg/ml solution of BSA. After another repeated washing, the beads wereresuspended to a suitable concentration in a suitable buffer and storedrefrigerated until use.

Phosphorylcholine with a linker arm can also be conjugated to KLH(keyhole limpet hemocyanin) via a diazophenyl group. More preferably ap-nitrophenyl-6-(O-phosphocholine)hydroxyhexanoate derivative of PC canbe synthesized according to Chesebro, B. and Metzger, H. (1972) Biochem.11:776. p-Nitrophenyl-6-(O-phosphocholine) hydroxyhexanoate wasdissolved in dry acetonitrile (100 mg/ml) just prior to adding it to theKLH. Derivative and KLH were mixed overnight at 4° C. and then dialyzedto remove unbound spacer and p-nitrophenylate, which is the leavinggroup.

An injection solution of the prepared phosphorylcholine conjugate,suspended in a suitable buffer, can be directly used for immunization.

Immunization with a Phosphorylcholine Conjugate

A high titer of IgM antibodies recognizing phosphorylcholine wasdetermined in plasma from BALB/c mice after immunization with 200 μg[p-Nitrophenyl-6-(O-phosphocholine) hydroxyhexanoate—KLH] i.p. using thesuggested immunoassay method.

Monoclonal Antibodies Against a Phosphorylcholine Conjugate

Monoclonal antibodies can be produced using any standard method known inthe art. See for example “Briles D E, Forman C, Hudak S. Claflin J L.Anti-phosphorylcholine antibodies of the T15 idiotype are optimallyprotective against Streptococcus pneumoniae. J Exp Med. 1982;156:1177-85” or “T15 PC binding monoclonal antibodies retain specificitywhen they switch from IgM to IgG., Spira, Gad; Aguila. Hector L.;Scharff, Matthew D. Fac. Med., Techniton-Israel Inst. Technol., Haifa,Israel. Journal of Immunology (1988), 140(8), 2675-80.

Other antibodies against a phosphorylcholine conjugate can be preparedusing methods well known to those skilled in the art. For example, asubfraction with aPC activity of a human immunoglobulin preparation canbe prepared, for example as described below, for example by affinitypurification using a phosphorylcholine conjugate. Intravenousimmunoglobulin preparations (e.g. IGIV; Baxter and others) is a highlypurified preparation of IgG commercially available and is used in thetreatment of patients who have no, or very low levels of antibodyproduction. Immunoglobulin preparations include those available from thefollowing manufacturers: Baxter (US) eg Gammagard®, Isiven (AntimoNaples, Italy). Omrix (Tel-Hashomer, Israel), Miles (Biological ProductsDivision, West Heaven, Conn.), Sclavo (Lucca, Italy), Sandoz (Novartis,Basel, Swizerland) eg Sandoglobulin®, Biotest Diagnostic Corporation(Deville, N.J.). Examples of immunoglobulin preparations are GammagardS/D®, Gammar IV®, Gammar-P IV®, Gammimune N®, Iveegam®, Panglobulin®,Polygam S/D®, Sandoglobulin®, Venoglobulin®. Immunoglobulin preparationstypically contain some IgM as well as IgG. Trace amounts of IgM arepresent in Gammagard®. Pentaglobin (Biotest) is an enriched IgMpreparation which has been used for treatment of SARS. The subfractionwith aPC activity may comprise both IgG and IgM, or may be selected tocomprise mainly IgG (for example by starting with an IgG-richpreparation such as Gammagard® and/or by selecting for IgG); or mainlyIgM (for example by starting with an IgM-rich preparation such asPentaglobin and/or by selecting for IgM).

An antibody preparation with specificity to a phosphorylcholineconjugate binds to unconjugated phosphorylcholine and may also bind tophosphorylcholine (PC) present in PC-containing compounds in which PC isexposed, for example in lysophosphatidylcholine (lysoPC; see, forexample, Kim et al, J Exp Med. 2002 Sep. 2; 196(5):655-65). Thus, anantibody preparation with specificity to a phosphorylcholine conjugatemay also bind to lysphosphatidylcholine.

IgM Immunoglobulin Levels in Atherosclerotic Subjects

IgM autoantibody levels against phosphorylcholine in subjects withhypertension (diastolic pressure>95 mmHg) were determined at baselineand after 4 years in a correlation study of risk factors foratherosclerosis. The results are summarized below.

Carotid plaques were detected in 77 subjects (35%) at enrolment, and in84 subjects (38%) at the 4-year follow-up. In total 218 human subjectswere in the study. Increases in intima-media thickness (IMT) atfollow-up were less prevalent in subjects having high serum levels ofIgM to PC (75^(th) or 90^(th) percentile) at the time of enrolment.There is a significant difference between mean values in IgManti-phosphorylcholine antibody levels between individuals withincreased and decreased IMT (638.8±219.6 vs. 734.8±266.9, p=0.004).

The relationships between IgM autoantibodies to PC and changes in IMTwere independent of age, smoking habits, treatment with atenolol orlacidipine and blood lipids. IgM autoantibodies were also independent ofIgG values.

One embodiment of the present invention is thus to use aphosphorylcholine conjugate for the preparation of a pharmaceuticalcomposition to be used in the treatment or prevention ofatherosclerosis. The conjugate can be phosphorylcholine linked to aprotein or to a polymer. The pharmaceutical composition is preferablygiven by injection.

The proposed method of active immunization will modulate theautoantibodies titer which in turn will have a positive effect on thedevelopment of atherosclerosis.

Another embodiment of the invention is to use an antibody preparation,for example a monoclonal antibody, recognizing a phosphorylcholineconjugate for the preparation of a pharmaceutical composition to be usedin the treatment or prevention of atherosclerosis. The monoclonalantibody can be produced using methods known in the art.

A further embodiment of the invention is to provide a method ofdiagnosing the presence or absence of antibodies, for example IgM or IgGantibodies, towards phosphorylcholine which factor is related to anincreased or decreased risk of developing ischemic cardiovasculardiseases, using a phosphorylcholine conjugate. A preferred method is animmunoassay. The method may be used in assessing the patient's risk ofdeveloping or progression of ischemic cardiovascular disease.

FIGURES

FIG. 1a : Inhibition of antibody (IgM) binding to ELISA-plates coatedwith PC albumin by β2GPI, PS and CL. Inhibition by different antigens ofbinding to PC albumin-coated plates. In order to investigate thespecificity of aPC, competition assays were performed as described inthe Experimental section. Results are presented as mean±SD.

FIG. 1b : Inhibition of antibody (IgG) binding to ELISA-pates coatedwith PC albumin by (β2GPI, PS and CL. Inhibition by different antigensof binding to PC albumin-coated plates. In order to investigate thespecificity of aPC, competition assays were performed as described inthe Experimental section. Results are presented as mean±SD.

FIG. 2a : Inhibition of antibody (IgM) binding to ELISA-pates coatedwith PC albumin by oxLDL and MDA-LDL. Inhibition by different antigensof binding to PC albumin-coated plates. In order to investigate thespecificity of aPC, competition assays were performed as described inthe Experimental section. Results are presented as mean±SD.

FIG. 2b : Inhibition of antibody (IgG) binding to ELISA-pates coatedwith PC albumin by oxLDL and MDA-LDL. Inhibition by different antigensof binding to PC albumin-coated plates. In order to investigate thespecificity of aPC, competition assays were performed as described inthe Experimental section. Results are presented as mean±SD.

FIGS. 3a-3b : effect on oxLDL uptake in macrophages by aPC extractedfrom IGIV

We tested two groups: macrophages with oxLDL and macrophages withoxLDL+preincubation with aPC extracted from IVIG.

macrophage+-oxLDL (total 107 cells):

Weak staining 37/107=34.58%

Strong Staining 10/107=9.35%

total staining positive 47/107=43.93%

macrophage+Dil-oxLDL+aPC-group (checked 156 cells):

Weak staining 37/156=23.72%

Strong Staining 2/156=1.28%

total staining positive 39/156=25%.

FIG. 3A shows staining with Dil-labelled oxLDL. FIG. 3B shows stainingwith unlabeled oxLDL.

FIG. 4: Effect of pre-incubation of high antiphospholipid antibodies(aPLs) titer serum with human pooled immunoglobulin Gammagard® onAnnexin V binding to human umbilical endothelia cells (HUVECs): flowcytometry analysis after 24 hrs culture.

IVIG pre-incubated Median fluorescence intensity with serum at (MFI) ofAnnexin V binding 0 mg/ml 649 2.5 mg/ml 913 5 mg/ml 1269 10 mg/ml 1382

FIG. 5: effect of aPC on ICAM-induction in endothelial cells 1 ug/ml ofPAF was added to cultures of endothelial cells, with or withoutpreincubation with aPC IgM. Expression of ICAM-1 was tested by FACScan.Green line represents PAF effect, while red is PAF+aPC IgM and blackcontrol. The data clearly indicate a shift to the left of histogram whenaPC IgM are added.

EXPERIMENTAL Subjects

Serum samples were obtained from 226 subjects with establishedhypertension (diastolic pressure >95 mm Hg) prior to their entry intothe Swedish component of the European Lacidipine Study onAtherosclerosis (ELSA)^(25,26). Samples were collected following a4-week washout period with no medication to minimize the effects oftreatment on the measured parameters. Blood pressure, cholesterol andtriglyceride levels were determined as described previously^(25,26). Onehundred and fifteen of the subjects were subsequently assigned totreatment with the β-blocker atenolol, and 111 of the subjects wereassigned to treatment with the calcium antagonist lacidipine. The studywas approved by the Ethics Committee of the Karolinska Hospital and wasconducted in accordance with the Helsinki Declaration. All subjects gaveinformed consent.

Carotid Ultrasound

Carotid ultrasound determinations were performed and analysed asdetailed elsewhere 25,26. A total of 218 patients had valid ultrasoundmeasurements at baseline and 4-year follow up. Briefly, the right andleft carotid arteries were examined with Biosound 2000 IIA duplexscanner using an 8.0 MHz annular array transducer. The intima-media(I-M) thickness was determined in the far wall as the distance betweenthe leading edge of the lumen-intima echo and the leading edge of themedia-adventitious echo. The outcome measurement as a surrogateindicator for atherosclerosis was the change in mean maximumIntimal-Medial thickness (IMT) of the four far walls in the distalcommon carotids and carotid bifurcations bilaterally (CBMmax) at the4-year follow-up. The associations between antibody levels to PC atenrolment into the study with an increase or decrease in IMT at the4-year follow-up were evaluated.

Reagents

Polysorp F96 microtiter immuno-plates were purchased from Nunc (RoskildeDenmark), PC-BSA (Phosphorylcholine-Bovine Serum Albumin) was purchasedfrom Biosearch Technologies, INC (USA).

Bovine serum albumin (BSA), Alkaline phosphatase conjugated goatanti-human IgG (r-chain specific). Alkaline phosphatase conjugated goatanti-human IgM (u-chain specific), PNPP (Alkaline phosphatasesubstrate), were obtained from Sigma (St. Louis, Mo., USA). Cardiolipin(CL) was purchased from AVANTT (US, β₂ glycoprotein (β₂GP1) was obtainedfrom Calbiochem (US).

Total IgG and IgM levels were determined by routine techniques aspreviously described⁶.

CRP was analyzed in serum by highly sensitive methods usingparticle-enhanced immunonephelometry (Behring Nephelometer Analyzer, BNII (Dade Behring GmBH, Marburg, Germany)) with an anti-assay variation<4%.

Determination of Autoantibodies Against PC, oxLDL and MDA-LDL

IgG and igM antibodies to PC-BSA were determined by enzyme-linkedimmunosorbent assay (ELISA). Pooled serum from 17 antiphospholipidsyndrome patients was used as an internal standard and tested on everyplate. The plateau of antibody binding was reached with the antigenconcentration of 10 μg/ml. F96 microtiter polysorbate was thereforecoated with PC-BSA (10 μg/ml) 50 μg·well in PBS. Coated plates wereincubated overnight at 4° C. After five washings with PBS, the plateswere blocked with 2% BSA-PBS for 2 h at room temperature and washed asdescribed above. Serum samples were diluted (1:30) in 0.2% BSA-PBS andadded at 50 μl/well.

LDL was isolated from plasma of healthy donors by sequential preparativeultra-centrifugation and oxidized by use of copper ions (OxLDL) orderivatized with MDA (MDA-LDL) as described⁶.

OxLDL and MDA-LDL were determined by ELISA essentially as described⁶.OxLDL or MDA-LDL was diluted to 2 μg/ml in coating buffer(carbonate-bicarboatne buffer 50 mM pH9.7), and 100 μl/well was used tocoat ELISA plates (Costar 2581). The plates were kept at 4° C.overnight, washed 4 times with PBS, and then blocked with 20% adultbovine serum in PBS (20% ABS-PBS) for 2 hours in room temperature. Theywere then incubated with 100 μl serum, diluted 1:30 in 20% ABS-PBS at 4°C. overnight.

Plates were incubated overnight at 4° C. and washed as described above.Alkaline phosphatase conjugated goat anti-human IgG (diluted 1:9000 inthe sample buffer) and Alkaline phosphatase conjugated goat anti-humanIgM (diluted 1:7000 in the sample buffer) were added at 100 μl/well andincubated at 4° C. overnight. After five washing, color was developed byadding the alkaline phosphatase substrate (PNPP) at 100 μl/well andincubating the plates for 60 min at room temperature in the dark. Theplates were read in an ELISA Multiskan Plus spectrophotometer at 405 nm.All samples were measured in a single assay and the coefficient ofvariation was below 10-15%.

Specificity of Anti-Phosphorylcholine-BSA Antibodies

In order to investigate the specificity of anti-phosphorylcholine-BSA,absorption assays were performed by use of pooled high titer sera. At adilution giving 50% of maximal binding to PC-BSA, high titer pooled serawere preincubated with different concentration of PC-BSA. Aftervortexing, the tubes were incubated at 40° C. overnight and centrifugedat 13000 r.p.m. for 30 min(40° C.) The supernatants were tested forantibody binding to PC-BSA as described. The percentage of inhibitionwas calculated as follows:

Percent inhibition=(OD without competitor−OD with competitor)×100/ODwithout competitor.

Statistical Analysis

anti-phosphorylcholine levels were dichotomized at the 75th and 90thpercentile. The association between anti-phosphorylcholine (or otherantibodies) and the progression of atherosclerosis over a 4-year periodwere determined by estimating increases in IMT (yes or no) usinglogistic regression analysis and the calculation of odds ratios (ORs)and 95% confidence intervals (CI), or comparison using Spearmancorrelation as indicated. Adjustments were made for possible cofoundersincluding age, smoking habits, serum cholesterol, serum triglyceridesand mode of anti-hypertensive treatment (lacidipine, atenolol). Atwo-tailed p-value <0.05 was considered as significant.

Results

Basic characteristics of the subjects at the time of enrolment into thestudy have been detailed elswhere (Pockley et al (2003) Hypertension 42,235-238) and are presented in Table 1.

Competition studies reveal that aPC of IgM and IgG subclass was competedout by preincubation with PC-BSA, while cardiolipin had a weak andphosphatidylserine no competitive capacity (FIG. 1a, 1b ).β2-glycoprotein I competed to some extent with IgG binding to PC-BSA butnot so much with IgM (FIG. 1a, 1b ). PC-BSA had a low capacity tocompete out binding to the other antigens tested (data not shown). OxLDLand MDA-LDL could compete out binding of IgM aPC to PC-BSA, and also ofIGG aPC, though not to the same extent (FIG. 2a, 2b ).

Increases in IMT at follow-up were less prevalent in subjects havinghigh serum levels of IgM to PC (75th or 90th percentile), oxLDL andMDA-LDL (90th percentile) at the time of enrolment, while CRP was notassociated with IMT-changes (Table 2).

Logistic regression analysis revealed that the relationships between IgMautoantibodies to PC, oxLDL and MDA-LDL and changes in IMT wereindependent of age, smoking habits, treatment with atenolol orlacidipine and blood lipids. aPC IgM were significantly associated withchanges in IMT at both 75th and 90th percentile, while aOxLDL andaMDA-LDL of IgM subclass only showed significance at the 90th (table3a-d). IgM autoantibodies were also independent of IgG values (data notshown). Furthermore, total IgG and IgM levels were not associated withIMT-measurements or changes (data not shown).

IgG autoantibodies to PC were trendwise lower in subjects with increasesin IMT but this difference did not reach statistical significance (Table2).

There were striking differences between men and women. aPC, aMDA-LDL,and aOxLDL of IgM subclass, were significantly higher in women than inmen (p's<0.05). In contrast, there were no differences between men andwomen in IgG levels of these autoantibodies. In addition, women had asignificantly lower occurrence of plaque at baseline and follow-up(p<0.05).

aPC IgM levels correlated negatively with increase in IMT (Rho 0.18,p=0.006) in contrast to two other protection factors, HDL and HSP70which did not correlate with IMT changes as continuous measurements(data not shown). Unlike aPC IgM. aOXLDL and aMDA-LDL did not reachsignificance in these determinations (data not shown).

There were significant associations between aPC IgM levels and aOCLDLIgM (Rho 0.74, p<0.001) and aMDA-LDL IgM (rho 0.51, p<0.001). LikewiseaPC correlated with HSP60 (Rho 0.28, p<0.001), HSP70 (Rho 0.35,p<0.001), which we recently described as a novel protective a factor forhuman atherosclerosis in this cohort (Pockley et al (2003) supra) andalso with HDL (Rho 0.23, p<0.01). There were not associations betweenaPC IgM, aOxLDL IgM or aOxLDL, MDA-LDL and LDL, CRP or triglycerides(data not shown).

When separate logistic regression analyses were made for men and women,controlling for age, total cholesterol, triglycerides, smoking andtreatment IgM aPC showed significant protective effects in women onlywhen 90th percentile was studied (EXP (B)=0.17, 95% CI=0.05-0.68; p=0.01and in men only when 75th percentile was studied EXP (B)=0.18, 95%CI=0.04-0.74; p=0.01, respectively).

IgM to MDA-LDL and oxLDL differend in this respect, since only valuesfor women reached statistical significance independently. Thus, whenseparate logistic regression analyses were made for men and women,controlling for age, total cholesterol, triglycerides, smoking andtreatment both IgM to MDA-LDL and IgM to OxLDL showed significantprotective effects in women (EXP (B)=0.17, 95% CI=0.05-0.68, p=0.01 andEXP (B)=0.18, 95% CI=0.04-0.74, p=0.01, respectively), whereas theeffect did not reach significance among men (EXP (B)=0.60, 95%CI=0.15-2.2, p=0.44, and EXP (B)=0.39, 95% CI=0.10-1.5, p=0.17respectively), indicating that high IgM titers to OxLDL and to MDA-LDLmay be specifically protective among women.

TABLE 1 Basic characteristics of the study group at enrolment. Resultsare presented as means (SD) or percentage (%) and mg/dL for lipids.Total Atenolol Lacidipine (N = 226) (N = 115) (N = 111) Age (years) 57.7(7.8) 57.6 (7.6) 57.7 (7.9) Sex (% males) 50 46 53 BMI 26.7 (3.7) 26.3(3.3) 27.1 (3.9) Total cholesterol 232.4 (37.8) 233.5 (38.1) 231.4(37.4) HDL  55.6 (27.6)  56.5 (25.8)  54.7 (27.6) LDL 149.4 (37.8) 149.7(37.1) 149.2 (38.6) Trigyclerides 131.6 (58.2) 128.6 (57.0) 134.7 (59.5)

TABLE 2 Unadjusted prediction of changes in IMT with baseline levels ofIgG and IgM autoantibodies to phosphorylcholine (PC). (95% CI) VariableOdds Ratio Lower Upper P 75th percentile aPC (IgG) .60 .32 1.1 .10 aPC(IgM) .46 .25 .85 .01 aOxLDL 1.2 .64 2.3 .57 (IgG) aOxLDL .77 .41 1.4.40 (IgM) aMDA-LDL .80 .43 1.5 .48 (IgG) aMDA-LDL .67 .36 1.2 .18 (IgM)C-reactive .80 .43 1.5 .46 protein 90th percentile aPC (IgG) .60 .25 1.4.24 aPC (IgM) .36 .15 0.87 .024 aOxLDL .94 .38 2.31 .90 (IgG) aOxLDL .27.11 .69 .006 (IgM) aMDA LUL .63 1.5 .30 .30 (IgG) aMDA-LDL .27 .11 .69.006 (IgM) C-reactive .60 .24 1.4 .24 protein

TABLE 3a prediction of changes in MT over a 4-year period using the 75thpercentile of aPC IgM autoantibodies at baseline in subjects withestablished hypertension. Variable Coeffi- Estimated in the cient oddsratio 95% CI model (B) Exp(B) P Lower Upper Smoking −.01 .99 .95 .66 1.5Sex −.05 .95 .87 .54 1.4 Total .003 1.0 .45 .99 1.0 cholesterol Plasma−.001 .99 .63 .99 1.0 tri- glycerides Age (years) .01 1.0 .59 .97 1.0Treatment −.23 .79 .40 .45 1.4 (A/L) APC IgM −1.0 .37 .0027 .15 .89

TABLE 3b prediction of changes in IMT over a 4-hyear period using the90th precentile of aPC IgM autoantibodies in subjects with establishedhypertension. Variable Coeffi- Estimated in the cient odds ratio 95% CImodel (B) Exp(B) P Lower Upper Smoking −.02 .97 .90 .65 1.5 Sex −.0051.0 .98 .56 1.8 Total .003 1.0 .42 .99 1.0 cholesterol Plasma −.001 0.99.67 .99 1.0 tri- glycerides Age (years) .003 1.0 .87 .97 1.0 Treatment−.22 .80 .43 .46 1.4 (A/L) aPC IgM −.77 .46 .017 .24 .87

TABLE 3c Prediction of changes in IMT over a 4-year period using the90th percentile with IgM autoantibodies to OxLDL and other risk factorsin subjects with established hypertension Variable Coeffi- Estimated inthe cient odds ratio 95% CI model (B) Exp(B) P Lower Upper Smoking −.01.99 .95 .66 1.5 Sex .001 1.1 .98 .56 1.8 Total .001 1.0 .72 .99 1.1cholesterol Plasma −.001 1.0 .71 .99 1.0 triglycerides Age (years) .011.0 .60 .97 1.1 Treatment −.28 .77 .35 .44 1.3 (A/L) aOxLDL IgM −1.3 .76.008 .11 .72

TABLE 3d Predicition of changes in IMT over a 4-year period using the90th percentile with IgM autoantibodies to MDA-LDL and other riskfactors in subjects with established hypertension Variable Coeffi-Estimated in the cient odds ratio 95% CI model (B) Exp(B) P Lower UpperSmoking −.07 0.93 .73 .62 1.4 Sex −.001 .99 .99 .56 1.7 Total 0.001 1.0.78 .99 1.0 cholesterol Plasma −.001 .99 .74 .99 1.1 triglycerides Age(years) 0.01 1.0 .54 .97 1.0 Treatment −2.7 .76 .34 .44 1.3 (A/L)aMDM-LDL -1.1 .31 .01 .12 .79 IgM

REFERENCES

-   1. Frostegard J, Ulfgren A K, Nyberg P, Hedin U, Swedenborg J,    Andersson U, Hansson G K. Cytokine expression in advanced human    atherosclerotic plaques: dominance of pro-inflammatory (Th1) and    macrophage-stimulating cytokines. Atherosclerosis. 1999; 145:33-43.-   2. Binder C J, Chang M K, Shaw P X, Miller Y I, Hartvigsen K, Dewan    A, Witztum J L. Innate and acquired immunity in atherogenesis. Nat    Med. 2002; 8:1218-26.-   3. Frostegard J. Autoimmunity, oxidized LDL and cardiovascular    disease. Autoimmun Rev. 2002; 1:233-7.-   4. Palinski W, Miller E, Witztum J L, Immunization of low density    lipoprotein (LDL) receptor-deficient rabbits with homologous    malondialdehyde-modified LDL reduces atherogenesis. Proc Natl Acad    Sci USA. 1995; 92:821-5.-   5. Xu Q, Dietrich H, Steiner H J, Gown A M, Schoel B, Mikuz G,    Kaufmann S H, Wick G. Induction of arteriosclerosis in    normocholesterolemic rabbits by immunization with heat shock    protein 65. Arterioscler Thromb. 1992; 12:789-99.-   6. Wu R, de Faire U, Lemne C. Witztum J L, Frostegard J.    Autoantibodies to OxLDL are decreased in individuals with borderline    hypertension. Hypertension. 1999; 33:53-9.-   7. Hulthe J, Wiklund O, Hurt-Camejo E, Bondjers G. Antibodies to    oxidized LDL in relation to carotid atherosclerosis, cell adhesion    molecules, and phospholipase A(2). Arterioscler Thromb Vase Biol.    2001; 21:269-74.-   8. Karvonen J, Paivansalo M, Kesaniemi Y A, Horkko S. Immunoglobulin    M type of autoantibodies to oxidized low-density lipoprotein has an    inverse relation to carotid artery atherosclerosis. Circulation.    2003; 108:2107-12.-   9. Bergmark C. Wu R, de Faire U, Lefvert A K, Swedenborg J. Patients    with early-onset peripheral vascular disease have increased levels    of autoantibodies against oxidized LDL. Arterioscler Thromb Vase    Biol. 1995; 15:441-5.-   10. Salonen J T, Ylä-Herttuala S, Yamamoto R, Butler S, Korpela H,    Salonen R, Nyyssönen K, Palinski W, Witztum J L. Autoantibody    against oxidised LDL and progression of carotid atherosclerosis.    Lancet. 1992; 339:883-887.-   11. Svenungsson E, Jensen-Urstad K. Heimburger M, Silveira A,    Hamsten A, de Faire U, Witztum J L, Frostegard J. Risk factors for    cardiovascular disease in systemic lupus erythematosus. Circulation.    2001; 104:1887-93.-   12. Frostegard J, Wu R, Giscombe R, Holm G, Lefvert A K, Nilsson J.    Induction of T-cell activation by oxidized low density lipoprotein.    Arterioscler Thromb. 1992; 12:461-7.-   13. Stemme S, Faber B, Holm J, Wiklund O, Witztum J L, Hansson G K.    T lymphocytes from human atherosclerotic plaques recognize oxidized    low density lipoprotein. Proc Natl Acad Sci USA. 1995; 92:3893-7.-   14. Berliner J A, Territo M C, Sevanian A, Ramin S, Kim J A, Bamshad    B, Esterson M, Fogelman A M. Minimally modified low density    lipoprotein stimulates monocyte endothelial interactions. J Clin    Invest. 1990; 85:1260-6.-   15. Frostegard J, Nilsson J, Haegerstrand A, Hamsten A, Wigzell H,    Gidlund M. Oxidized low density lipoprotein induces differentiation    and adhesion of human monocytes and the monocytic cell line U937.    Proc Natl Acad Sci USA. 1990; 87:904-8.-   16. Frostegard J, Haegerstrand A, Gidlund M, Nilsson J. Biologically    modified LDL increases the adhesive properties of endothelial cells.    Atherosclerosis. 1991; 90:119-26.-   17. Fei G Z, Huang Y H, Swedenborg J, Frostegard J. Oxidised LDL    modulates immune-activation by an IL-12 dependent mechanism.    Atherosclerosis. 2003; 169:77-85.-   18. Bochkov V N, Kadl A, Huber J, Gruber F, Binder B R, Leitinger N.    Protective role of phospholipid oxidation products in    endotoxin-induced tissue damage. Nature. 2002; 419:77-81.-   19. Frostegard J, Huang Y H, Ronnelid J, Schafer-Elinder L.    Platelet-activating factor and oxidized LDL induce immune activation    by a common mechanism. Arterioscler Thromb Vase Biol. 1997;    17:963-8.-   20. Heery J M, Kozak M, Stafforini D M, Jones D A, Zimmerman G A,    McIntyre T M, Prescott S M. Oxidatively modified LDL contains    phospholipids with platelet-activating factor-like activity and    stimulates the growth of smooth muscle cells. J Clin Invest. 1995;    96:2322-30.-   21. Subbanagounder G, Leitinger N, Shih P T, Faull K F, Berliner    J A. Evidence that phospholipid oxidation products and/or    platelet-activating factor play an important role in early    atherogenesis: in vitro and In vivo inhibition by WEB 2086. Circ    Res. 1999; 85:311-8.-   22. Harnett W, Harnett M M. Phosphorylcholine: friend or foe of the    immune system? Immunol Today. 1999.20:125-9.-   23. Shaw P X, Horkko S, Chang M K, Curtiss L K, Palinski W,    Silverman G J, Witztum J L. Natural antibodies with the T15 idiotype    may act in atherosclerosis, apoptotic clearance, and protective    immunity [see comments]. J Clin Invest. 2000; 105:1731-40.-   24. Binder C J, Horkko S, Dewan A, Chang M K, Kieu E P, Goodyear C    S, Shaw P X, Palinski W, Witztum J L, Silverman G J. Pneumococcal    vaccination decreases atherosclerotic lesion formation: molecular    mimicry between Streptococcus pneumoniae and oxidized LDL. Nat Med.    2003; 9:736-43.-   25. Zanchetti A, Bond M G, Hennig M, Neiss A, Mancia G, Dal Palu C,    Hansson L, Magnani B, Rahn K H, Reid J, Rodicio J, Safar M, Eckes L,    Ravinetto R. Risk factors associated with alterations in carotid    intima-media thickness in hypertension: baseline data from the    European Lacidipine Study on Atherosclerosis. J Hypertens. 1998;    16:949-61.-   26. Zanchetti A, Bond M G, Hennig M, Neiss A, Mancia G, Dal Palu C,    Hansson L, Magnani B, Rahn K H, Reid J L, Rodicio J, Safar M, Eckes    L, Rizzini P. Calcium antagonist lacidipine slows down progression    of asymptomatic carotid atherosclerosis: principal results of the    European Lacidipine Study on Atherosclerosis (ELSA), a randomized,    double-blind, long-term trial. Circulation. 2002; 106:2422-7.

Study Showing Protective Effect of aPC

In an observational study from Malmo (the Malmo Diet and Cancer Study),about 6000 out of 30000 subjects from the cohort were recruited forextensive cardiovascular investigations, including non-invasiveassessment of subclinical atherosclerosis through ultrasoundmeasurements of the carotids. In addition, additional cardiovascularrisk factors were measured at baseline. These subjects have beenfollowed for over 10 years with regard to the occurrence of new eventsof cardiovascular diseases (myocardial infarction, chronic coronaryheart disease, atherotrombotic stroke). In order to assess the relativerisks (calculated as relative hazards) with 95% confidence intervals,nested-case-control analyses (3 controls per case) were undertaken forlow levels of antibodies against phosphorylcholine (aPC-IgM). There werein total 145 CVD cases (mainly myocardial infarction (MI) and ischemicstroke) and 400 age and sex-matched controls. The cutoff level for aPCwas 307 for the tenth percentile of aPC levels. There were in total 20CVD cases with aPC levels below the tenth percentile (14%), and 34controls (9%), corresponding to a relative hazard of 1.9 (95% CI1.1-4.3). The corresponding number of male CVD cases below the tenthpercentile of aPC was 16 (19%), and 25 control patients below this level(11%), corresponding to a relative hazard of 1.9 (95% CI 1.1-3.5). Thenumber of female cases was too low to yield robust information onrelative risks (see Tables 1 and 2). The results suggest that low aPClevels are predictive for the occurrence of cardiovascular disease inhealthy subjects, and could act as markers for cardiovascular diseases.

TABLE 1 Descriptive statistics for aPC (<10^(th) percentile) SEX MalesFemales All Below 10^(th) pct Case Control Case Control Case Control No68 206 57 160 125 366 % 81 89 93 95 86 92 Yes N 16 25 4 9 20 34 % 19 117 5 14 9

TABLE 2 Univariate analysis of the influence of aPC (<10^(th)percentile) on CVD by conditional logistic regression for all patients,males and females, respectively. Hazard 95% Hazard Ratio Variablep-value ratio Confidence Limits All patients aPC 0.0308 1.939 1.0633.536 Males aPC 0.0262 2.181 1.097 4.338 Females aPC 0.6556 1.331 0.3794.676

Effects of aPC Introduction

By use of columns which are preabsorbed with PC-BSA, PC-KLH orPneumococcal vaccine (Statens Serum Institute, Denmark), we extractantibodies with reactivity against these compounds. Levels of aPC IgGare raised in at least the two first of these. Small amounts of IgM maybe extracted out of IVIG and then also run through the columnspreabsorbed with the abovementioned antigens. Through this method we canobtain polyclonal human aPC of IgG and IgM subclass. Protein measurementindicates that aPC IgM levels of 0.5 mg/ml could be extracted. Usingthese antibodies we can test their functional properties using in vitromodels:

-   -   1. Can preincubation of increasing concentrations of aPC IgM        with oxidized LDL decrease binding and uptake in        monocyte/macrophage cell line, THP1? Test systems with confocal        microscopy and/or FACS can be used.    -   2. Can preincubation of increasing concentrations of aPC IgM        with normal IgM as control, with PAF, lysophosphatidylcholine        (LPC) inhibit induction of adhesion molecules ICAM on        endothelial cells by these lipids? Also other cytokines can be        tested using a commercial kit (several different cytokines;        BioSource). Tests can make use of FACScan.

Cell Culture

Cryopreserved pooled HUVECs at passage 2 were purchased from CascadeBiologics, Inc. (Portland, Oreg., USA). Cultures were maintained in EGM™phenol red-free medium (Clonetics, San Diego, Calif., USA), containing2% of fetal bovine serum and supplements. The cells were incubated in 75cm² flasks (TPP, AG, Trasadingen, Switzerland) at 37° C. underhumidified 5% CO₂ conditions.

All experiments were performed at passage 3 to 4. Cells were seeded at2×10⁴ cells/ml density in 12-well plates (NUNC, Inc, Naperville, Ill.,USA) for flow cytometry analysis. After allowing 12-24 hours forattachment the cells were made quiescent in SFM for at least 12 hrsprior to treatment.

Monocytic cell line THP-1 was from AT&T (USA). Cells were maintained inRPMI with 10% FCS.

Preparation of aPC

Total IgM or IgG fraction was separated from commercially availablepooled human immunoglobulin (Gammagard®) at 50 mg/ml using HiTrap IgM orIgG columns (Amersham Biosciences). Antibodies against phosphorylcholine(PC) were eluted after loading IgM or IgG fraction on NHS-Sepharosecolumns coupled to PC conjugated either to keyhole limpet protein(KLH)(1 or 5 mg/ml) or to bovine serum albumin (BSA) (1 mg/ml), followedby BSA-only column. PC-BSA (Phosphorylcholine-Bovine Serum Albumin) andPC-KLH was purchased from Biosearch Technologies, INC (Ca, USA). Elutedfractions were buffer-exchanged on PD-columns and concentrated withMillipore Centricone® devices. Procedures were performed according toinstructions given by manufacturers. The concentration of IgM aPCprepared was typically 50 μg/ml, and the concentration of IgG aPC wastypically 30 μg/ml.

Scavenger Binding and Uptake of oxLDL by THP-1 Derived Macrophages

Oxidized LDL (oxLDL) is prepared as described by incubation with copperions. First, oxLDL is labeled with Dil(Dil-(1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanineperchlorate; Molecular Probes, Inc)) and diluted in Saline-EDTA bufferto 1 mg/ml. After that, 2 ml lipoprotein-deficient serum is added for 1mg of oxLDL and then filtered (0.45 um). 50 ul Dil (3 mg/ml) in DMSO isadded for 1 mg oxLDL and the mixture is incubated 15 h, 37° C. and thendialyzed against several changes of saline-EDTA for 6 h. After this themixture is 0.45 um filtered again.

Uptake of the oxLDL is studied with fluorescence/confocal microscopy.THP-1 cells as models for monocytes/macrophages are grown overnight onslide chamber. (medium: DMEM/10% FBS/Glu/PEST)

3× wash with DMEM medium without FBS

Incubated with oxLDL-DiO 5 ug/ml (SFM medium) 6 h.

The cells washed with 0.2% BSA-PBS 5×. PBS 1×

Macrophage nucleus staining: the cells were incubated with 1 ug/mlbisbenzimide 10′ and wash with PBS 3×.

Fix and mount: the cells were then fixed with 4% paraformaldehyde in PBSfor 30′, PBS 3×, finally, after 1 drop of mounting gel. The slides werecovered with cover slip.

Annexin V Binding to Endothelial Cells

Heparin-preserved plasma with high capacity to inhibit Annexin V bindingwas added to HUVECs monolayer at concentration of 10% in SFM. After 24hrs cells were harvested with Cell Dissociation Solution (CDS;Sigma-Aldrich, St. Louis, Mo., USA) and carefully pooled withsupernatants, to exclude selective loss of detached floating cells,centrifugation at 1200 rpm for 7 min followed. After resuspension in 100μl of annexin V-binding buffer (Molecular Probes Inc, Eugene, Oreg.,USA) samples were stained with 2 μl 5 mg/ml annexin V-FITC (MolecularProbes) and incubated for 15 min on ice. Shortly before acquisition 1mg/ml of propidium iodide (PI; a vital dye; R&DSystems Europe Ltd,Abingdon, UK) was added. Analysis was performed as described above.

Statistical Analysis

The statistics were computed using Stat View software, SAS Institute AB,Göteborg, Sweden. Skewed continuous variables were logarithmicallytransforme. Study groups were compared using ANOVA for continuousvariables and Chi square for categorical variables. Fischer's PLSD wasused as post hoc test. Correlation coefficients were calculated usingSimple regression or for not normally distributed variables Spearman'srank correlation. The significance level was set at p<0.05.

Results Measurements of Annexin V Binding to Endothelial Cells

The frequency of HUVECs positive for annexin V staining was determinedeither as percentage of annexin V⁺/PI⁻ cells on a bivariate dot plot orpercentage of annexin V⁺ cells based on a histogram. Annexin V-bindingto HUVEC's in the presence of serum known to decreased binding andpreincubated with IVIG was determined. Preincubation with IVIG couldrestore binding of Annexin, indicating that antibodies present in IVIGcould neutralize binding (FIG. 4). APC-BSA and aPC-KHL were bothassociated significantly in SLE-patients with a history of CVD withAnnexin V binding to EC (r=0.45; p=0.02 and r=0.42 and p=0.03respectively). aPC were determined as described above.

Effect on oxLDL Uptake in Macrophages by aPC

aPC of IgM and IgG subclass, extracted from IVIG as indicated werepreincubated with oxLDL indicated (FIG. 3). We used total IgM as controlfor aPC IgM (macrophage+Dil-oxLDL+IgM) and effect on macrophage uptake.The total percentage of positive staining cells is 46.62%, indicatingthat IgM per se does not have the inhibitory effects that aPC has. IgMwas bought from SIGMA, and is purified human IgM is produced byprecipitation and gel filtration techniques using normal human serum asthe starting material. The immunoglobulin is determined to be at least95% pure.

Effect of aPC on ICAM-Induction in Endothelial Cells

PAF was incubated with EC at the indicated concentrations. Asdemonstrated in FIG. 5 this lipid could induce a significant increase inICAM-expression. aPC of IgM subclass, extracted from IVIG as indicatedwere preincubated with these lipids as indicated (FIG. 5).

Correlations Between aPC and Other Risk Markers in ELSA Study (226Individuals with Hypertension as Described Previously

aPC IgM was associated with two other protection factors, HSP 70 andHDL, as indicated in Table 4. There was also a weak albeit significantassociation with TNF, a marker of inflammation and a proatherogeniccytokine.

TNF is an important pro-inflammatory cytokine and TNF levels negativelyassociated with aPC igM levels. The association is weak, butsignificant.

HSP 70 is a novel protection factor recently described by us and others.There is a clearly positive association. Also HSP60, which is a weakerprotection factor, is associated.

HDL is a well known “good” cholesterol, with anti-inflammatoryproperties. It is associated significantly with aPC IgM.

ANTPCIGG ANTPCIGM Spearman's ANTPCIGG Correlation .000 .245 rhoCoefficient Sig. (2-tailed) .000 N 220 220 ANTPCIGM Correlation .2451.000 Coefficient Sig. (2-tailed) .000 N 220 220 HDL Correlation .008.233 Coefficient Sig. (2-tailed) .906 .001 N 206 206 TNFA Correlation−.012 −.136 Coefficient Sig. (2-tailed) .863 .044 N 220 220 HSP60Correlation .138 .279 Coefficient Sig. (2-tailed) .047 .000 N 209 209HSP70 Correlation .157 .356 Coefficient Sig. (2-tailed) .022 .000 N 213213 ** Correlation is significant at the .01 level (2-tailed). *Correlation is significant at the .05 level (2-tailed).

1-16. (canceled)
 17. A method for assessing a human patient's risk ofdeveloping or progression of cardiovascular disease comprising: (a)providing phosphorylcholine (PC) conjugated to an immunologic carrier(IC), (b) contacting said PC/IC conjugate with an antibody-containingsample from said patient, (c) detecting antibodies from said samplecomplexed with PC/IC conjugate, thereby determining the level of PC/ICconjugate-reactive antibodies in sample, and (d) diagnosing said patientas having an increased risk of developing or progression ofcardiovascular disease when a low level of PC/IC conjugate-reactiveantibodies is determined.
 18. The method of claim 17, wherein thecardiovascular disease is ischemic cardiovascular disease.
 19. Themethod of claim 17, wherein the cardiovascular disease isatherosclerosis.
 20. The method of claim 17, wherein the level of IgMantibodies from said sample complexed with the PC/IC conjugate isdetermined.
 21. The method of claim 17, wherein the level of IgGantibodies from said sample complexed with the PC/IC conjugate isdetermined.
 22. The method of claim 17, wherein the PC is linked to saidIC via a spacer.
 23. The method of claim 17, wherein the IC is aprotein, lipid, polymer or latex bead.
 24. The method of claim 23,wherein the protein is keyhole limpet hemocyanin (KLH) or human serumalbumin (HSA).
 25. The method of claim 17, wherein the PC/IC conjugatecomprises phosphorylcholine linked to a latex bead, optionally via aspacer.
 26. The method of claim 17, wherein detecting comprises anenzyme linked immunosorbent assay.
 27. The method of claim 17, whereinsaid patient exhibits hypertension.